The mechanisms by which IL-4 mediates signal transduction events in interleukin-3 (IL-3)-dependent myeloid lines which respond mitogenically to IL-4 were further elucidated. IL-4 was demonstrated to induce pronounced tyrosine phosphorylation and activation of a 170-kDa substrate, designated 4PS, in FDC-P1 and FDC-P2 cell lines. The 4PS substrate was found to be weakly related immunologically to IRS-1, the principal substrate tyrosine phosphorylated by insulin in cells of connective tissue origin. Insulin (and IGF-1) evoked a potent mitogenic response in these cells and also phosphorylated and activated 4PS, indicating these related growth factors utilize similar signaling pathways in hematopoietic cells. The IL-3-dependent 32D cell line, unlike FDC-P1 and FDC-P2 lines, do not respond mitogenically to IL-4 or insulin and do not express 4PS. Transfection of an expression vector containing IRS-1 into the 32D line restored IL-4- and insulin-mediated mitogenesis. When vectors containing IL-4 or insulin receptors were simultaneously expressed with IRS-1, 32D cells could grow long term in the respective ligands. These results provide evidence that IRS-1 (or possibly 4PS) expression is essential for IL-4- or insulin-induced mitogenesis and long-term growth is dependent on IRS-1 expression coupled with a sufficient number of growth factor receptors. The mechanisms by which the bovine papillomavirus E5 protein induces transformation are under investigation. The IL-3-dependent 32D cell line was transfected with an expression vector containing the E5 gene. While E5 expression had no transforming effect on 32D cells, dual expression of E5 with the beta platelet-derived growth factor receptor abrogated IL- 3 dependence and induced a tumorigenic phenotype.